Note: Descriptions are shown in the official language in which they were submitted.
ANTI-EGFR/HIGH AFFINITY NK-CELLS COMPOSMONS AND METHODS FOR CHORDOMA TREATMENT
[00011 This application claims priority to copending US Provisional
Application with the
serial number 62/504,689, which was filed May 11, 2017.
Field of the Invention
[00021 The field of the invention is modified immune competent cells for the
treatment of
diseases, especially as it relates to high affinity natural killer (haNK)
cells and anti-EGFR
compositions for treatment of chordoma.
Background of the Invention
100031 The background description includes infonnation that may be useful in
understanding
the present invention. It is not an admission that any of the information
provided herein is
prior art or relevant to the presently claimed invention, or that any
publication specifically or
implicitly referenced is prior art.
100041 All publications and patent applications herein are incorporated by
reference to the
same extent as if each individual publication or patent application were
specifically and
individually indicated to be incorporated by reference. Where a definition or
use of a term in
an incorporated reference is inconsistent or contrary to the definition of
that term provided
herein, the definition of that term provided herein applies and the definition
of that term in
the reference does not apply.
100051 Chordoma is a rare bone tumor and is thought to be derived from the
residual
notochord. Accounting for 20% of primary spinal tumors (1-4% of all malignant
bone
tumors), about 300 new cases per year are diagnosed in the United States, with
approximately
2400 patients alive with chordoma in the U.S. The median overall survival from
time of
diagnosis is an estimated 6-7 years. Surgery followed by radiation therapy is
the usual
"standard of care," but the anatomic location and size of the tumor often
prevent curative
excision with clear margins. Thus, relapse is common and metastases have been
reported in
up to 40% of cases. No agent has been approved by the U.S. Food and Drug
Administration
for chordoma therapy since it is largely resistant to standard cytotoxic
chemotherapy, creating
an urgent need for novel therapeutic modalities for chordoma.
1
Date Recue/Date Received 2021-08-12
100061 More recently, new treatment regimens have been proposed based on
various
molecular profiles of chordoma. For example, miRNA was proposed to
downregulate EGFR
as described in PLoS One (2014); 9(3): e91546. Combined inhibition of IFG-1R
and EGFR
showed durable response in one trial (Front Oneol (2016); 6:98), while various
small
molecule inhibitors of EGFR such as erlotinib, gefitinib, lapatinib,
sapitinib, or afatinib were
described as potential therapeutic agents based on in vitro data in J Pathol
(2016); 239: 320-
334.
100071 In other examples, an immune therapeutic approach targeting PD-Li
expressed on
chordoma cells using avelumab (anti-PD-L1 antibody) was employed as described
in
Oncotarget (2016);7(23):33498-511. While conceptually elegant, various
difficulties
nevertheless remain. Among other things, PD-Li is also expressed on various
non-chordoma
cells and as such off-target ADCC may occur. Moreover, even under in vitro
condition using
1FN-gamma stimulation and normal donor NK cells, avelumab mediated ADCC was
relatively low (about 25-35% lysis of all targeted chordoma cells). In still
further known
approaches, chordoma cells lines were irradiated in vitro with low dose
ionizing radiation to
increase EGFR expression and were then exposed to cetuximab (anti-EGFR
antibody).
Subsequent exposure to normal donor NK cells indicated someADCC (see Abstract
FASEB
Journal, Vol. 31, No.1 Suppl; Abstract No. 934.12: Exploiting Immunogenic
Modulation in
Chordoma: Sublethal Radiation Increases EGFR Expression and Sensitizes Tumor
Cells to
Cetuximab). However, radiation is often not well tolerated and ADCC activity
without
radiation was less than desirable. Therefore, most of the more recent attempts
to treat
chordoma were less than successful or have not resulted in a regimen approved
by regulatory
agencies.
100081 Thus, while various treatment methods and compositions for chordoma are
known in
the art, all or almost all of them suffer from one or more disadvantages.
Thus, there remains a
need for improved compositions and methods for treatment of chordoma.
Summary of The invention
100091 The inventive subject matter is directed to compositions, kits, and
methods of
treatment of chordoma that includes co-administration of haNK cells with an
anti-EGFR
antibody to so trigger ADCC (antibody dependent cell-mediated cytotoxicity)
and augment
EGFR-based treatments. Most notably, therapeutic effect is attained not by way
of
2
Date Recue/Date Received 2021-08-12
interference with EGFR signaling, but via NK cell (and especially high-
affinity NK cell)
mediated cytotmdc cell killing. Thus, suitable anti-EGFR antibodies may be
agonistic or
antagonistic, or may elicit no signaling change in response to binding, and
preferred NK cells
will have a CD16 variant with a binding affinity to the Fe portion on an IgG
that is above the
affinity of a wild type CD16 (e.g., 158FF).
100101 Therefore, in one aspect of the inventive subject matter, the inventor
contemplates a
method of treating chordoma that includes a step of co-administering an anti-
EGFR antibody
and a high affinity NK (haNK) cell to a patient in need thereof at a dosage
effective to treat
the chordoma. Most preferably, it is contemplated that the anti-EGFR antibody
is a
monoclonal antibody with binding specificity against human EGFR, and/or that
the anti-
EGFR antibody is an IgG1 to so trigger ADCC. Therefore, viewed from a
different
perspective, it is contemplated that the anti-EGFR antibody may be a humanized
non-human
anti-EGFR antibody, and most preferably is cetuximab.
100111 With respect to administration it is generally contemplated that the
anti-EGFR
antibody is administered at a dosage of between 100 mg/m2 and 1,000 mg/m2,
preferably at
the same time as the haNK cell. Thus, the anti-EGFR antibody may also be bound
to a high-
affinity CD16 that is expressed on a surface of the haNK cell. Contemplated
haNK cells are
preferably administered at a dosage of between 5x105 cells/kg and 5x108
cells/kg, and it is
further preferred that the haNK cells are a NK92 derivative and/or (typically
intracellularly)
express recombinant IL2. Moreover, it is generally preferred that the haNK
cell is genetically
engineered to have a reduced expression of at least one inhibitory receptor
and/or that the
haNK cell is genetically engineered to express a CD16 158V variant.
100121 Where desired, contemplated methods may further include a step of
administering a
further cancer treatment to the patient, most typically an immune therapy
(e.g.,
administration of a recombinant yeast or recombinant virus expressing a
patient- and tumor-
specific ncoepitope, or administration of a recombinant yeast or recombinant
vinis expressing
brachyury) and/or chemotherapy (e.g., administration of irinotecan,
gemcitabine,
capecitabine, 5-FU, FOLFIRI, FOLFOX, and/or oxiplatin). Moreover, suitable
further cancer
treatments may also comprise radiotherapy.
100131 Therefore, the inventor also contemplates a pharmaceutical composition
that includes
an anti-EGFR antibody that is coupled to a high affinity variant of CD16,
wherein the CD16
3
Date Recue/Date Received 2021-08-12
high affinity variant is expressed on the surface of a genetically engineered
NK cell. With
respect to the antibody and the genetically engineered cell, the same
considerations as above
apply. In addition, it is generally preferred that the pharmaceutical
compositions will be
formulated for transfusion and comprise between 1x106 cells and 5x109 cells.
100141 Therefore, the inventors also contemplate a pharmaceutical kit that
comprises an anti-
EGFR antibody and a plurality of a high affinity NK (haNK) cells. Once more,
with respect
to the antibody and the genetically engineered cell, the same considerations
as above apply.
In view of the above, it should therefore be recognized that the inventors
also contemplate the
use of a high affinity NK (haNK) cell to augment treatment of chordoma wherein
the
treatment comprises administration of an anti-EGFR antibody.
100151 Various objects, features, aspects and advantages of the inventive
subject matter will
become more apparent from the following detailed description of preferred
embodiments,
along with the accompanying drawing figures in which like numerals represent
like
components.
Brief Description of the Drawing
100161 Figure lA is a schematic illustration of a treatment based on anti-EGFR
and haNK
cells.
100171 Figure 1B is a table listing frequencies of allelic variants and
binding affinity for
CDI6 Fe receptors in human donor cells and in genetically engineered haNK
cells.
100181 Figure 2 depicts various graphs for selected phenotypes of CD16
polymorphism-
genotyped NK cells and haNK cells.
100191 Figure 3 is a graph depicting exemplary results for EGFR expression in
selected
chordoma cell lines.
100201 Figure 4 is a graphical representation of exemplary results for in
vitro assays for
ADCC activity mediated by cetuximab relative to an isotype control antibody.
100211 Figure 5 is a graphical representation of exemplary results for in
vitro assays for
ADCC activity mediated by cetuximab using FCGR3A (CD16 gene)-genotyped normal
donor NK cells that expressed the FcgRIIIa (CD16)-158 FF, VF, or VV allele.
4
Date Recue/Date Received 2021-08-12
100221 Figure 6 is a graphical representation of exemplary results in which
cetuximab
increased haNK-cell lysis via ADCC in selected chordoma cell lines at two
different time
points indicating multiple cell killing by haNK cells.
[0023] Figure 7 is a graphical representation of exemplary results for
affinity of cetuximab to
CD16 of selected NK cells versus haNK cells.
100241 Figure 8 is an exemplary treatment schema for Induction Phase as
contemplated
herein.
100251 Figure 9 is an exemplary treatment schema for Maintenance Phase as
contemplated
herein.
Detailed Description
100261 The inventor has now discovered that chordoma can be effectively
treated using
haNK cells in combination with an anti-EGFR antibody (e.g., cetuximab) to so
induce in a
patient an ADCC response/NK cytotoxic cell killing with desirable therapeutic
effect as is
exemplarily depicted in Figure 1A. Such treatment may be implemented prior to,
and/or
concurrent with radio- and/or chemotherapy, and/or may be employed with immune
therapy
as is discussed in more detail below.
100271 It should be noted that the antibodies contemplated herein are not used
as an EGFR
signaling inhibitor, but as an target specific beacon for a natural killer
cell, and most
preferably a high-affinity NK cell (haNK) to facilitate binding of the CD16
receptor of the
NK cell to the Fe portion of the bound antibody and so to eradicate the tumor
cell via
ADCC/NK cytotoxic cell killing. With respect to the high affinity cells it
should be
appreciated that the high affinity may be due to patient idiosyncratic
mutations at the CD16
locus (which may be hetero- or homozygous and occur at relatively low
frequency), and more
typically may be due to genetic engineering of NK cells to express a high
affinity variant
(e.g., F158V) from a recombinant nucleic acid. Therefore, it is typically
preferred that the
treatment includes combined administration of an anti-EGFR antibody and high
affinity NK
cells. Such administration may be performed sequentially, with the antibody
being
administered in a first step and the NK cells being transfused in a second
subsequent step
(e.g., within 24 hours of administration of the antibody), or simultaneously
where the anti-
EGFR antibody is bound to the CD16 receptor of the high affinity NK cell.
Date Recue/Date Received 2021-08-12
100281 Therefore, in one preferred example, the inventor now contemplates that
chordoma
treatment with an anti-EGFR antibody can be significantly improved by co-
administration of
the anti-EGFR antibody with a genetically modified NK cell that expresses a
high affinity
CD16 variant (and where the NK cell most preferably also expresses
intracellularly IL-2).
Notably, due to the high affmity of the CD16 variant to the constant region of
the antibody,
tight binding and activation of the NK cell is achieved using the binding
specificity of the
anti-EGFR antibody to the EGFR of the tumor cell. Thus, it should be noted
that
contemplated treatments advantageously compensate for the most common, low
affinity,
variants of CD16 that is present in a large proportion of human (at least
70%). Figure 1B
depicts allele frequencies for CD16. Viewed from a different perspective, use
of genetically
modified NK cells will allow for an increase in ADCC in patients even where
the patients
have a low affinity CD16 (158F/F) phenotype. On the other hand, it is also
contemplated that
patients may also be identified as having a high-affinity CD16 (158VN)
phenotype. Such
patients may then receive the anti-EGFR antibody without, or with a lower
total dosage of
haNK cells (e.g., between 104-106 cells or between 105-107 cells per
transfusion).
100291 With respect to suitable anti-EGFR antibodies it is contemplated that
such antibodies
may vary considerably in origin, sequence, and serotype. However, it is
generally preferred
that the anti-EGFR antibody will have a constant region (Fe) that binds with
high affinity to
the CD16 variant. Thus, and most typically, the constant region is a constant
region of a
human IgG land the CD16 variant is a 158VN variant. However, it should be
appreciated that
suitable CD16 variants and constant region variants may be specifically
tailored to the
specific antibody and/or a specific subset of genetically modified NK cells.
As will be
readily appreciated, high affinity pairs (CD16 variant/ constant region
variant) can be
identified using numerous manners known in the art, and especially preferred
manners
include affinity maturation via phage display, RNA display, two-hybrid library
screening
using CD16 variant as bait and constant region library as prey (or vice
versa), etc. Likewise,
known high-affinity antibodies may be subject to CDR grafting (with the CDRs
being
specific towards EGFR) to so obtain a high-affinity anti-EGFR antibody.
100301 Moreover, it should be recognized that while commercially available
EGFR
antibodies such as cetuximab and panitumumab are especially preferred, other
contemplated
anti-EGFR antibodies include monoclonal antibodies with binding specificity
against human
EGFR, and especially IgGi type antibodies that are humanized non-human anti-
EGFR
6
Date Recue/Date Received 2021-08-12
antibodies. There are numerous commercially available anti-EGFR antibodies
known in the
art (e.g., from ABCAM, Millipore, Biolegend, etc.), and all of them are deemed
suitable for
use herein. Additionally, suitable anti-EGFR antibodies may also include EGFR
binding
fragments that are coupled (preferably covalently as chimeric protein) to a
CD16 binding
domain (or domain variant).
100311 For example, suitable anti-EGFR antibodies include clinically approved
cetuximab
and paninunumab, as well as human and non-human antibodies such as ab52894,
ab131498,
ab231, ab32562, ab32077, or ab76153 (all commercially available from Abcam,
USA), as
well as AY13 (Biolegend, USA) and 06-847 (Millipore, USA). These antibodies
may be used
directly, or in humanized form, or CDR regions may be grafted onto a human
IgG. Likewise,
suitable CDRs for grafting can be found in U5584409 and WO 2011/156617.
100321 With respect to suitable NK cells it is generally contemplated that the
NK cells may
be autologous NK cells from the patient, and such autologous NK cells may be
isolated from
whole blood, or cultivated from precursor or stem cells using methods known in
the art.
Moreover, it should be appreciated that the NK cells need not be autologous,
but may also be
allogenic or heterologous NK cells. Still further, it is contemplated that the
NK cells may be
HLA matched NK cells, which may be primary cells, NK cells differentiated from
upstream
stem or progenitor cells, or cultured NK cells. However, in particularly
preferred aspects of
the inventive subject matter, the NK cells are genetically engineered to
achieve one or more
desirable traits, and particularly preferred NK cells are NK92 cells, or
derivatives of NK92
cells. Consequently, suitable NK cells will also be continuously growing
(Immortalized')
cells. For example, in one particularly preferred aspect of the inventive
subject matter, the
genetically engineered NK cell is a NK92 derivative that expresses 1L-2
(typically in an
intracellularly retained, non-secreted manner) and is modified to have reduced
or abolished
expression of at least one inhibitory receptor (MR), which renders such cells
constitutively
activated (via lack of or reduced inhibition).
100331 For example, suitable NK cells may have one or more modified MR that
are mutated
such as to reduce or abolish interaction with MHC class I molecules. Of
course, it should be
noted that one or more KIRs may also be deleted or expression may be
suppressed (e.g., via
miRNA, siRNA, etc.). Most typically, more than one KIR will be mutated,
deleted, or
silenced, and especially contemplated KIR include those with two or three
domains, with
short or long cytoplasmic tail. Viewed from a different perspective, modified,
silenced, or
7
Date Recue/Date Received 2021-08-12
deleted KIRs will include KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A,
KIR2DL5B, KIR2DS1, KIR2DS2, K1R2DS3, KIR2DS4, KIR2DS5, IUR3DL1, KIR3DL2,
KIR3DL3, and KIR3DS1. Such modified cells may be prepared using protocols well
known
in the art. Alternatively, such cells may also be commercially obtained from
NantKwest (see
URL www.nantkwest.com) as aNK cells ('activated natural killer cells).
100341 In a particularly preferred aspect of the inventive subject matter, the
NK cell is a
genetically engineered NK92 derivative that is modified to express a high-
affinity Fey
receptor (CD16). Sequences for high-affinity variants of the Fey receptor are
well known in
the art (see e.g., Blood 2009 113:3716-3725), and all manners of generating
and expression
are deemed suitable for use herein. Expression of such receptor is believed to
advantageously
increase specific targeting and cytotoxic cell killing of tumor cells when
using antibodies that
are specific to a patient's tumor cells. Viewed from a different perspective,
contemplated
anti-EGFR antibodies will provide exquisite targeting specificity against
chordoma cells
while such genetically engineered NK92 derivative have high affinity to
antibodies where the
antibodies have bound to the cognate antigen, and further have significantly
increased
cytotoxic killing ability in the context of antibody binding. Of course, it
should be
appreciated that such targeting antibodies are commercially available and can
be used in
conjunction with the cells (e.g., bound to the Fey receptor). Likewise, such
genetically
engineered NK92 derivative cells may also be commercially obtained from
NantKwest as
haNK cells ('high-affinity natural killer cells).
100351 In further contemplated embodiments, the NK cells will be irradiated
before
transfusion to prevent continuous cell division. While not limiting to the
inventive subject
matter, the cells will typically be irradiated that abrogates cell division,
but that still allows
fort metabolic activity, and NK cell function (especially cytotoxic cell
killing). Therefore,
suitable radiation dosages for the NK cells will be between 50 cGy and 2,000
cGy.
Furthermore, such radiation is typically beta or gamma radiation, however,
other manners
such as e-beam irradiation are also expressly contemplated herein.
100361 Most typically, both the anti-EGFR antibody and the high affinity NK
(haNK) cells
are administered to the patient using dosages and routes that are known in the
art for
administration of both, antibodies and NK cells. Therefore, suitable dosages
for
administration of the anti-EGFR antibody (e.g., cetuximab) will typically be
between 100
mg/m2 and 1,000 mg/m2, or between 100 mg/m2 and 300 mg/m2, or between 300
mg/m2 and
Date Recue/Date Received 2021-08-12
600 mg/m2, or between 600 mg/m2 and 900 mg/m2, or even higher. Administration
is
preferably intravenous over a period of between about 1 min and 120 min, and
more typically
between about 10 min and 60 min. Likewise, haNK cells are preferably
administered at
dosages suitable for cell transfusions. Therefore, suitable dosages will
typically be in the
range of between 5x105 cells/kg and 5x108 cells/kg, and most typically between
5x106
cells/kg and 5x107 cells/kg. Administration is preferably intravenous over a
period of
between about 1 min and 120 min, and more typically between about 10 min and
60 min.
100371 In further contemplated aspects of the inventive subject matter, the
administration of
the anti-EGFR antibody and the haNK. cells is preferably contemporaneous such
that both the
anti-EGFR antibody and the haNK cells are present in the patient's blood in
measurable
quantities at the same time. Consequently, co-administration of the anti-EGFR
antibody and
the haNK cells may be performed at the same time, or within 10 minutes or
within 30
minutes or within 2 hours of each other. Moreover, it should also be
appreciated that upon
and/or during administration the anti-EGFR antibody may be non-covalendy bound
to the
haNK cells via the CD16 variant.
100381 Based on preclinical evidence of the role of EGFR in chordoma
pathogenesis and the
observation by irrununohistochemistry that over 70% of chordoma specimens
express EGFR,
several clinical trials targeting EGFR have previously been undertaken in
chordoma.
However, because these trials were not randomized or well controlled, no
consensus had been
reached concerning the therapeutic benefit of EGFR inhibition in chordoma. In
two separate
case reports, the combination of the EGFR MAb cetuximab and gefitinib, a
tyrosine kinase
inhibitor of EGFR, achieved partial radiographically defmed responses. Here,
and as shown
in more detail below, the inventor demonstrates that cetuximab, when combined
with haNK
cells, markedly and significantly increased NK cell based lysis, and
especially lysis via
ADCC.
100391 Some previous clinical studies have also shown that FcgRIlla
polymorphisms of NK
cells correlated with response to IgGi MAb therapy. Notably, patients with
metastatic breast
cancer who had FCGR2A-131 HH and/or FCGR3A-158 VV genotypes had a
significantly
better objective response rate and progression-free survival with trasturtunab
therapy than
patients with neither genotype. Similarly, in a study of 49 patients with
follicular lymphoma,
FCGR3A-158 VV patients had an improved response to rituximab. Three
retrospective
9
Date Recue/Date Received 2021-08-12
studies in metastatic colorectal cancer patients treated with cetuximab
reported that VV is the
most beneficial FCGR3A-158 genotype.
100401 Although ADCC induction can be observed in in vitro models, clinical
translation
often raises various obstacles. First, recruiting sufficient numbers of
functionally active NK
cells to tumor tissues is technically challenging since they often represent
only 10% of
lymphocytes, and are frequently dysfunctional in a cancer-induced
inununosuppressive
environment. Moreover, first-line treatment for metastatic/advanced chordoma
(i.e.,
chemotherapy and radiation therapy) is also very likely to reduce the number
and activity of
lymphocytes. Independently, adoptive NK-cell therapies have been developed to
supply
sufficient numbers of functional NK cells for patients. The cytotoxic NK-92
cell line was
generated for adoptive transfer therapy from a 50-year-old male patient with
progressive non-
Hodgkin's lymphoma. Four phase I trials in different malignancies have been
conducted
using irradiated NK-92 cells. The infusions were well tolerated, and clinical
responses were
observed in patients with hematological malignancies, melanoma, lung cancer,
and kidney
cancers. However, since NK-92 cells do not express the FcgRilIa receptor, they
cannot
mediate ADCC. In contrast, genetically engineered cells expressing a high-
affinity CD16a,
Vi 58 FcyRIIIa receptor have now been established and are also commercially
available (e.g.,
as haNK cells from NantKwest, 9920 Jefferson Blvd., Culver City, CA 90232).
100411 Since only approximately 14% of the population is homozygous for the
high-affinity
FcgRIlla receptor (FCGR3A-158 VV), the inventor contemplates infusing haNK
cells into
patients who carry the genotype of low- or intermediate-affinity FcgRITIa
receptor to
maximize MAb efficacy. Among other things, and as shown in more detail below,
the
inventor noted that haNK cells have a 2.8-fold higher affinity to cetuximab
than NK cells
from healthy donors carrying FCGR3A-158 FF. Consistent with their high binding
ability to
cetuximab, haNK cells also significantly induced ADCC via cetuximab in
chordoma cells.
Moreover, since 109 to 1010 irradiated NK-92 cells were shown to be safely
administered to
cancer patients, the inventor contemplates levels of adoptive transfer of
irradiated haNK
cells, even in patients whose endogenous NK cells express the VV phenotype
(but possibly at
a lower total dosage, such as 80% or less, or 70% or less, or 50% or less, or
40% or less than
dosage administered to patient with 158FF phenotype).
100421 NK-92 cells have been shown to express large numbers of activating
receptors such as
NKp30, NKp46, and NKG2D. NKG2D and DNAM-1 are the best-characterized
activating
Date Recue/Date Received 2021-08-12
NK-cell receptors implicated in immune response against cancers. Both
receptors recognize
their ligands expressed on tumor cells and induce target-cell lysis. As shown
in more detail
below, haNK cells have higher expression of NKG2D and DNAM-1 compared to
normal NK
cells, indicating a greater ability to recognize and lyse tumor cells.
Notably, without
cetuximab, NK cells from normal (158FF phenotype) donors lysed chordoma cells
at
extremely low levels without cetuximab (data not shown). In contrast, haNK
cells induced
substantially greater lysis of chordoma cells, even without cetuximab.
100431 Consequently, it is contemplated that adaptively transferred irradiated
haNK cells will
provide sufficient numbers of functional NK cells for all chordoma patients
and could so
functionally 'convert' FCGR3A-158 FF carriers to VV carriers. Therefore, it
should be
appreciated that cetuximab plus irradiated haNK cell-mediated immunotherapy
may have
potential clinical benefit for patients with chordoma. Moreover, it should be
recognized that
while cetuximab is described as a suitable target, numerous additional or
alternative targets
are also deemed appropriate for use in conjunction with the teaching presented
herein. For
example, suitable targets include receptors and kinases that are preferably
selectively or
exclusively expressed at the cell surface of chordoma cells, and particularly
include MET,
PDGFR, and ERBB2. Moreover, where the chordoma cells have mutations that lead
to
neoepitopes in one or more proteins, it is contemplated that antibodies may be
prepared that
will bind to the neoepitope where the neoepitope is visible or presented on
the surface of the
cell.
100441 Of course, it should be appreciated that additional therapeutic
interventions may be
used with or complement contemplated treatments. For example, suitable
treatments include
radiation and/or chemotherapy using agents such as irinotecan, gemcitabine,
capecitabine, 5-
FU, FOLFIRI, FOLFOX, and/or oxiplatin. In further contemplated aspects,
contemplated
treatments may also include immune modifiers such as IL15, IL15 superagonists,
interferon-
gamma to increase PD-L1 expression, and/or checkpoint inhibitors targeting
checkpoint
receptors and/or their ligands (e.g., PD-Li antibody (avelumab)).
100451 In addition, it is contemplated that immune therapy may also be based
on generation
of an immune response against brachyury. For example, immune therapy may be
performed
using recombinant viruses (and especially adenoviruses) that include a nucleic
acid segment
encoding brachyury (or a portion thereof). Infected cells, such as dendritic
cells, will then
express and process the recombinant protein for presentation as a MHC-I and/or
MHC-II
11
Date Recue/Date Received 2021-08-12
complex. In other aspects, heat-killed recombinant yeast may be genetically
modified to
express brachytuy with potential antineoplastic activity. Upon subcutaneous
administration,
the brachyury-expressing yeast vaccine is then recognized by dendritic cells,
processed, and
presented by Class I and II MI-IC molecules on the dendritic cell surface,
which is thought to
elicit a targeted CD4+ and CD8+ T-lymphocyte-mediated immune response.
Examples
100461 In Vitro Examples
100471 Cell culture and reagents: The chordoma cell lines JHC7 and UM-Chorl
were
obtained from the Chordoma Foundation (Durham, NC). The chordoma cell lines U-
CH2
(ATCCO CRL-3218 TM) and MUG-Chorl (ATCCO CRL-3219 TM) were obtained from
American Type Culture Collection (Manassas, VA). All cell lines were passaged
for fewer
than 6 months and were maintained as previously described (Oncotarget, 2016
May 9). haNK
cells were cultured in phenol-red free and gentamycin-free X-Vivo-10 medium
(Lonza,
Walkersville, MD) supplemented with 5% heat-inactivated human AB serum (Omega
Scientific, Tarzana, CA) at a concentration of 5x105/ml. haNK cells were
irradiated with 10
Gy 24 h before all experiments. Peripheral blood mononuclear cells (PBMCs)
from healthy
volunteer donors were obtained from the NIH Clinical Center Blood Bank
(NCT00001846).
100481 Flow cytometry: Antihuman MAbs used were as follows: PE-EGFR (BD
Biosciences,
San Jose, CA), FITC-CD16 clone 3G8 (BD Biosciences), APC-CD56 (BioLegend, San
Diego, CA), PE-CD226 (DNAM-1) (BD Biosciences), PerCP-Cy5.5-NKG2D (BD
Biosciences), PE-Cy7-perforin (eBioscience, San Diego, CA). Samples were
acquired on a
FACSCalibur flow cytometer or FACSVerse (Becton Dickinson, Franklin Lakes, NJ)
and
analyzed using FlowJo software (TreeStar, Inc., Ashland, OR). Isotype control
staining was <
5% for all samples analyzed.
100491 Antibody-dependent cellular cytotoxicitv assay: The ADCC assay was
performed as
known in the art, with indicated modifications. NK effector cells were
isolated from normal
donor PBMCs using the Human NK Cell Isolation (negative selection) Kit 130-092-
657
(Miltenyi Biotec, San Diego, CA) following the manufacturer's protocol,
resulting in > 80%
purity, and allowed to rest overnight in RPMI-1640 medium containing 10% fetal
bovine
serum. Tumor cells were harvested and labeled with 111In. Cells were plated as
targets at
2,000 cells/well in 96-well round-bottom culture plates and incubated with 10
WmL of
12
Date Recue/Date Received 2021-08-12
cetuxitnab (Erbitux6; Lilly, Indianapolis, IN) or irresponsive rituximab
(Rituxani); Biogen,
Cambridge, MA) as a control isotype antibody at room temperature for 30 min.
NK cells or
haNK cells were added as effector cells. Various effectortarget cell ratios
were used in the
study. After 4 h or 20 h, supernatants were harvested and analyzed for the
presence of "lin
using a WIZARD2 Automatic Gamma Counter (PerkinElmer, Waltham, MA).
Spontaneous
release was determined by incubating target cells without effector cells, and
complete lysis
was determined by incubation with 0.05% Triton X- 100 (Sigma-Aldrich, St.
Louis, MO).
Experiments were carried out in triplicate. Specific ADCC lysis was determined
using the
following equation: Percent lysis = [(experimental cpm ¨ spontaneous cpm) /
(complete cpm
¨ spontaneous cpm)] x 100. To verify that CD16 (FcgRIII) on NK cells engages
ADCC lysis
mediated by cetuximab, a CD16 MAb was used to block CDI6. NK cells were
incubated
with 2 pg/mL of CD16 MAb (clone B73.1; eBioscience) and haNK cells were
incubated with
50 iig/mL of CD16 MAb for 2 h before being added to target cells.
100501 CD16 (FcgRIIIa) genotyping: DNA was extracted from PBMCs of healthy
donors
using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA), and stored at ¨80 C
until use.
The polymorphism of CD16 at amino acid position 158 that is a valine (V) vs.
phenylalanine
(F) was determined using allele-specific droplet digital poly-merase chain
reaction (PCR)
employing the TaqMan array for CD16 (rs396991; Life Technologies, Waltham,
MA). A
master reaction mix was prepared, and 1 L of genotyping DNA was added. The
PCR
reaction was performed on a Bio-Rad T100 thermal cycler (Bio-Rad, Hercules,
CA) for 40
cycles at 95 C for 10 min, 94 C for 30 sec, and 60 C for! min. The plate was
read on a Bio-
Rad QX200 droplet reader. Data were analyzed with Bio-Rad QuantaSoft v.1.5
software.
[0051] Statistical analysis: Significant differences in the distribution of
data acquired by
ADCC assays were determined by paired Student's t test with a 2-tailed
distribution and
reported as P values, using Prism 6.0f software (GraphPad Software Inc., La
Jolla, CA).
[0052] The phenotype of CD16a polvmorphism-genotvped NK cells and haNK cells:
NK
cells from some individuals can be potent cytotoxic effectors for cancer
therapy. However,
there can be technical challenges to obtaining sufficient numbers of
functionally active NK
cells from patients. As an alternative, several cytotoxic NK cell lines have
been generated,
including NK-92. These NK-92 cells, designated haNK, have recently been
engineered to
endogenously express IL-2 and the high affinity (ha) CD16 V158 FcyRIIIa
receptor (haNK
cells, commercially available from NantKwest, 9920 Jefferson Blvd., Culver
City, CA
13
Date Recue/Date Received 2021-08-12
90232). The inventor compared the phenotype (CD56, DNAM-1, NKG2D, perforin,
and
CD16) of CD16a polymorphism-genotyped normal donor NK cells with that of haNK
cells.
100531 While there were only minor differences in the percentage of cells
expressing a given
marker, there were substantial differences observed in the levels of
expression as determined
by mean fluorescence intensity (MFI) as shown in the panels of Figure 2.
Compared to NK
VV donors, haNK cells had a 20-fold higher MFI of CD56 (Figure 2, Panel A),
2.9-fold
higher expression of DNAM-1 (Figure 2, Panel B), and 1.8-fold higher
expression of
NKG2D (Figure 2, Panel C). Notably, there was no difference in perforin
expression between
NK cells and haNK cells (Figure 2, Panel D), and the mean MFI of CDI6 was 1.5-
fold higher
in VV donors compared to FF donors and haNK cells (Figure 2, Panel E).
100541 It has previously been shown that chordoma cell lines express EGFR, and
the inventor
qualitatively confirmed and extended this finding, employing four human
chordoma cell
lines: JHC7, UM- Chorl, U-CH2, and MUG-Chorl with exemplary results shown in
Figure
3 (Inset numbers indicate "A positive cells and mean fluorescence intensity
(MFI)). As can be
seen, the four chordoma cell lines express between 13 /0 to 80% EGFR as
determined by flow
cytometry, although the absolute expression levels of EGFR can modulate with
tissue culture
density and time in culture.
100551 The inventor further performed an in vitro assay to determine cetuximab-
mediated
ADCC in chordoma cell lines employing NK cells from healthy donors as
effectors. As
shown in Figure 4, Panel A, cetuximab significantly increased NK-cell lysis
relative to the
isotype control antibody in JHC7 cells (13.7-fold; P <0.01), UM-Chorl cells
(10.5-fold; P <
0.01), U-CH2 cells (83.5-fold; P <0.01), and MUG-Chorl cells (59-fold; P
<0.01). Notably,
cetuximab alone (no NK cells) did not mediate lysis of chordoma cells (data
not shown). NK-
cell lysis via ADCC occurs when CD16 (FcgRIII) on NK effector cells interacts
with the Fe
portion of antibodies recognizing target cells. As shown in Figure 4, Panel B,
the addition of
CD16 neutralizing antibody inhibited cetuximab-enhanced NK-cell lysis in both
the IHC7
and UM-Chorl cell lines analyzed, indicating that cetuximab-induced NK-cell
lysis was
mediated by ADCC. More specifically, Panel A in Figure 4 depicts results for
ADCC assays
for four chordoma cell lines, using normal donor NK cells at an
effector:target (E:T) ratio of
20:1. Indicated groups were incubated with cetuximab. Panel B depicts results
for ADCC
assays with two chordoma cell lines, using normal donor NK cells at an E:T
ratio of 20:1.
Indicated groups were incubated with cetuximab and anti-CD16 antibody.
Statistical analyses
14
Date Recue/Date Received 2021-08-12
were done by Student's t-test, * = P < 0.05, error bars indicate mean S.D.
for triplicate
measurements. This experiment was repeated at least two times with similar
results.
100561 The inventor then performed in vitro assays for ADCC activity mediated
by
cetuximab using FCGR3A-genotyped normal donor NK cells that expressed the
FcgRIlia-
158 FF, VF, or VV allele. With control isotype antibody, UM-Chorl cells were
killed at very
low levels by NK cells regardless of NK phenotype as can be seen from the bar
graphs for all
allele types in Figure 5, Panel A. However, cetuximab increased NK-cell lysis
in all the NK-
cell phenotypes to varying degree: Cetuximab-induced lysis by NK cells from
three donors
expressing the FcgRIlIa-158 FF was 24%, 17%, and 15%, respectively. Notably,
cetuximab-
induced ADCC lysis by NK cells using three VF donors was 34%, 49%, and 32%,
respectively, and 51%, 66%, and 59% lysis, respectively, using NK cells from
three VV
donors. As can be seen from Panel B, there was a significant positive
correlation (R2= 0.85)
for the mean of cetuximab-mediated ADCC lysis induced by NK cells from three
FF (19%),
three VF (38%), three VV (59%) donors. Taken together, these results
demonstrate that NK
cells that express the FcgRIIIa-158 V allotype (as haNK cells express as well)
exhibit
significantly enhanced cetuximab-mediated ADCC in chordoma cells.
100571 To examine the potential utility of haNK cells for cetuximab therapy of
chordoma, the
inventor performed an in vitro assay for cetuximab-mediated ADCC using haNK
cells as
effectors (Figure 6A). Lysis by haNK cells with isotype control was 11.8%
ofJHC7 cells
and 2.6% of UM-Chorl cells. Cetuximab significantly enhanced haNK-cell lysis
compared to
isotype control in both JHC7 (1.7-fold; P <0Ø1) and UM-Chorl cells (2.6-
fold; P <0.01).
The addition of CD16 neutralizing antibody inhibited cetuximab-enhanced haNK-
cell lysis in
both jHC7 and UM-Chorl cell lines (data not shown). As NK cells have
previously been
shown to be "serial killers" (one NK cell can lyse up to five target cells),
20-h "In-release
assays were also carried out (Figure 6B). ADCC assays were performed using two
chordoma
cell lines, using haNK cells as effector cells at an E:T ratio of 20:1 for A.
4h and B. 20h.
Indicated groups were incubated with cetuximab and/or anti-CD16 antibody.
Statistical
analyses were done by Student's t-test, * = P < 0.05, error bars indicate mean
S.D. for
triplicate measurements. This experiment was repeated at least two times with
similar results.
100581 Here, the lysis of the two chordoma cell lines was markedly greater
after 20 hours as
compared to the 4 hour data in Panel A. These results indicate that haNK cells
induce
persistent ADCC via cetuximab in chordoma cells. To determine relative
affinities, the
Date Recue/Date Received 2021-08-12
inventor compared the ability of cetuximab to inhibit the binding of FITC-
conjugated CD16
MAb to CD16 polymorphism-genotyped normal donor NK cells and haNK cells
(Figure
7A). Remarkably, a 50% inhibition of CD16 Ab binding to NK cells from four FF
donors
was achieved with 220 pg/mL of cetuximab. Compared to FF donors, a 4.5-fold
lower (49.2
i.tg/mL) and 2.8-fold lower (80 Lig/mL) concentration of cetuximab showed a
50% inhibition
of CD16 Ab binding to normal NK cells from VV donor and haNK cells,
respectively
(Figure 7B). These results show that both NK cells expressing FcgRIIIa-158 VV
and haNK
cells bind cetuximab with higher affinity than NK cells expressing FcgRIlla-
158 FF. More
specifically, NK cells from four FF and two VV normal donors and haNK cells
(NantKwest,
9920 Jefferson Blvd., Culver City, CA 90232) were incubated with varying
concentrations of
cetuximab, followed by RTC-conjugated CD16 Ab. Percentages of inhibition of
CD16 MAb
binding were calculated as described above Panel A depicts percentages of
inhibition of
CD16 MAb binding shown by each donor. Panel B depicts the mean of percentages
of
inhibition of CD16 MAb binding.
100591 In Vivo Examples
[4:10601 In view of the above, numerous treatment protocols in vivo (typically
in human) are
contemplated that will preferably also include additional treatment regimens
or modalities
that will complement the targeted immune therapy using hanK cells and
cetuximab (or other
targeting antibody).
(00611 For example, one contemplated treatment will be administered in two
phases, an
induction and a maintenance phase, as described in more detail below.
Preferably, patients
will receive induction treatment for up to 1 year. Patients with complete
response (CR) in the
induction phase, ongoing stable disease (SD) or an ongoing partial response
(PR) at 1 year
will then proceed to the maintenance phase, and patients will remain in the
maintenance
phase for up to 1 year.
[0062] Tumors will be assessed at screening, and tumor response will be
assessed every 8
weeks in the first year or until complete response, and every 12 weeks in the
second year or
after a complete response by computed tomography (CT) or magnetic resonance
imaging
(Mu) of target and non-target lesions in accordance with Response Evaluation
Criteria in
Solid Tumors (RECIST) Version 1.1. For all patients, exploratory tumor
molecular profiling
will be conducted on samples collected during various time points (e.g., prior
to treatment, 8
16
Date Recue/Date Received 2021-08-12
weeks after the start of treatment, and during potential prolonged treatment
periods
(depending on response). Separate blood tubes will be collected every 6 weeks
in the first
year or until a complete response and every 8 weeks in the second year or
after a complete
response during routine blood draws for immunology and ctDNAJctRNA analyses.
100631 Contemplated exemplary treatment regimes will include a combination of
a vaccine
component, low dose metronomic chemotherapy (LDMC), cetuximab, NK cell
therapy, low-
dose radiation therapy, an 1L-15 superagonist, and a checkpoint inhibitor to
so maximize
immunogenic cell death (LCD) and to augment and maintain the innate and
adaptive immune
responses against cancer cells. More specifically, the treatment is designed
to interrupt the
escape phase of inununoediting by: (a) Mitigating potential immunosuppression
in the tumor
microenvoronment (TME), preferably by LDMC to reduce the density of Tregs,
MDSCs, and
M2 macrophages that contribute to immunosuppression in the TME; (b) Inducing
and
coordinating LCD signals, preferably via LDMC and low-dose radiation therapy
to increase
the antigenicity of tumor cells. Cetuximab and avelumab will be used to
enhance ADCC and
cytotoxic T-cell activity; (c) Conditioning dendritic and T cells, preferably
by cancer
vaccines and an IL-15 superagonist to enhance tumor-specific cytotoxic T-cell
responses; (d)
Enhancing innate immune responses, preferably using NK cell therapy (e.g., in
combination
with cetuximab) will be used to augment the innate immune system, and an IL-15
superagonist will be used to enhance the activity of endogenous and introduced
NK cells. (e)
Hypofractionated-dose radiation therapy to upregulate tumor cell NK ligands to
enhance
tumor cytotoxicity of NK cells; and maintaining immune responses. Checkpoint
inhibitors
will be used to promote long-term anticancer immune responses.
100641 To that effect, suitable agents included in the exemplary treatment are
summarized in
Table 1. It should therefore be recognized that by combining the agents that
simultaneously
target distinct but complementary mechanisms that enable tumor growth, the
treatment
regimen aims to maximize anticancer activity and prolong the duration of
response to
treatment. Moreover, the treatment will typically be administered in 2 phases:
an induction
phase and a maintenance phase. The purpose of the induction phase is to
stimulate immune
responses against tumor cells and mitigate immunosuppression in the TME. The
purpose of
the maintenance phase is to sustain ongoing immune system activity against
tumor cells,
creating durable treatment responses.
17
Date Recue/Date Received 2021-08-12
Agent Mitigating Inducing and Conditioning Enhancing
Maintaining
lmmunosuppression in Coordinating Dendritic Innate Immune
the TM E ICD Signals and T Cells Immune
Responses
Responses
Aldoxorubicin HCI X X
AL 1-803 X X X
Ad5-based vaccines: X
ETBX-051 (Brachyury)
and ETBX-061 (MUC1)
Yeast-based vaccine: X
GI-6301 (Brachyury)
haNK cells X
Avelurnab X
Cetuximab X
Cyclophosphamide X X
SBRT X X
Table 1
100651 Aldoxorubicin hydrochloride (HC1): Aldoxorubicin HC1 is an albumin-
binding
prodrug of the anticancer agent doxorubicin. Due to enhanced permeability of
the vasculature
within tumors, plasma albumin preferentially accumulates in solid tumors.
Aldoxorubicin
HC1 binds circulating albumin through a thiol reactive maleimide group
conjugated to the
doxorubicin molecule; binding to albumin results in targeting and accumulation
of the
aldoxorubicin HC1 prodrug in solid tumors. Doxorubicin has been postulated to
act through a
number of mechanisms including intercalation of DNA, inhibition of
topoisomerase
induction of apoptosis, inhibition of RNA synthesis, and/or interaction with
the cell
membrane. The chemical name for aldoxorubicin HCI is N-RE)-11-1(2S,4S)-4-
1(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yljoxy-2,5,12-trihydroxy-7-
methoxy-
6,11-dioxo-3,4-dihydro-1H-tetracen-2-y1]-2-hydroxyethylidenelamino]-6-(2,5-
dioxopyrrol-1-
y1)hexanamide; hydrochloride. Aldoxorubicin is manufactured by Baxter
Oncology.
18
Date Recue/Date Received 2021-08-12
100661 ALT-803 (recombinant human super agonist interleukin-15 (IL-15) complex
[also
known as IL 15N72DAL-15RaSu/IgG1 Fe complex]): ALT-803 is an IL-15-based
immunostimulatory protein complex consisting of two protein subunits of a
human IL-15
variant associated with high affinity to a dimeric human IL-15 receptor a (IL-
15Ra) sushi
domain/human IgG1 Fc fusion protein. The IL-15 variant is a 114 amino acid
polypeptide
comprising the mature human 1L-15 cytokine sequence, with an asparagine to
aspartate
substitution at position 72 of helix C (N72D). The human IL-15Ra sushi
domain/human IgG1
Fe fusion protein comprises the sushi domain of the human IL-15 receptor a
subunit (IL-
15Ra) (amino acids 1-65 of the mature human 1L-15Ra protein) linked to the
human IgG1
CH2-CH3 region containing the Fe domain (232 amino acids). Except for the N72D
substitution, all of the protein sequences are human. ALT-803 is manufactured
by Altor
Biosciences.
100671 ETBX-051 (Ad5 [El-, E2b-]-Brachyury vaccine): ETBX-051 is an Ad5-based
vector
that has been modified by the removal of the El, E2b, and E3 gene regions and
the insertion
of a modified hBrachyury gene. The modified hBrachyury gene contains agonist
epitopes
designed to increase cytotoxic T-Iymphocyte (CU) antitumor immune responses.
ETBX-051
is manufactured by Etubics.
100681 ETBX-061 (Ad5 [El-, E2b-]-mucin 1 [MUC,1] vaccine): ETBX-061 is an Ad5-
based
vector that has been modified by the removal of the El, E2b, and E3 gene
regions and the
insertion of a modified human MUC I gene. The modified MUC1 gene contains
agonist
epitopes designed to increase c-n, antitumor immune responses. ETBX-061 is
manufactured
by Etubics.
100691 G1-6301 (Brachy-ury yeast vaccine): GI-6301 is a heat-killed S.
cerevisiae yeast-based
vaccine expressing the hBrachyury oncoprotein. The Brachyury antigen is the
full-length
protein possessing an N-terminal MADEAP (Met-Ala-Asp-Glu-Ala-Pro) motif
appended to
the hBrachyury sequence to promote antigen accumulation within the vector and
a C-terminal
hexahistidine epitope tag for analysis by Western blotting. Expression of the
hBrachyury
protein is controlled by a copper-inducible CUP1 promoter. GI-6301 is
manufactured by
GlobeInunune.
100701 haNK1m, NK-92 [CD16.158V, ER IL-2], Suspension for Infusion (haNKTM for
Infusion): NK-92 [CD16.158V, ER IL-2] (high-affinity activated natural killer
cell line,
19
Date Recue/Date Received 2021-08-12
[haNKTM for Infusion]) is a human, allogeneic, NK cell line that has been
engineered to
produce endogenous, intracellularly retained 1L-2 and to express CD16, the
high-affinity
(158V) Fc gamma receptor (FcyRIIIa/CD16a). Phenotypically, the haNK cell line
is CD56+,
CD3-, and CD16+.
100711 The haNK cell line was developed by transfecting the parental activated
NK (aNK)
cell line (NK-92) with a bicistronic plasmid vector containing IL-2 and the
high-affinity
variant of the CD16 receptor. The plasmid contains an ampicillin resistance
cassette, and the
promoter used for expression of the transgene is elongation factor 1 alpha
with an SV40
polyadenylation sequence. The plasmid was made under transmissible spongifonn
encephalopathies-free production conditions and contains some human origin
sequences for
CD16 and 1L-2, neither of which have any transforming properties. haNKTM for
Infusion has
enhanced CD16-targeted ADCC capabilities as a result of the insertion of the
high-affinity
variant of the CD16 receptor. haNK cells are manufactured by NantK west.
100721 Avelumab (commercially available from Pfizer as BAVENCIO injection,
for
intravenous [IV] use): Avelumab is a human IgG1 lambda monoclonal antibody
directed
against the human immunosuppressive PD-L1 protein and has potential immune
checkpoint
inhibitory and antineoplastic activities. Avelumab has a molecular weight of
147 kDa. By
inhibiting PD-L1 interactions, avelumab is thought to enable the activation of
T cells and the
adaptive immune system. By retaining a native Fc-region, avelumab is thought
to engage the
innate immune system and induce ADCC.
100731 Cetuximab (commercially available from Eli Lilly as ERBITUX injection,
for IV
infusion): Cetuximab is a recombinant, human/mouse chimeric monoclonal
antibody that
binds specifically to the extracellular domain of human EGFR. Cetuximab is
composed of the
Fv regions of a murine anti-EGFR antibody with human IgG1 heavy and kappa
light chain
constant regions and has an approximate molecular weight of 152 kDa. Cetuximab
is
produced in mammalian (murine myeloma) cell culture. Cetuximab is a sterile,
clear,
colorless liquid of pH 7.0 to 7.4, which may contain a small amount of easily
visible, white,
amorphous cetuximab particulates. Cetuximab is supplied at a concentration of
2 mg/mL in
either 100 mg (50 mL) or 200 mg (100 mL), single-use vials. Cetuximab is
formulated in a
solution with no preservatives, which contains 8.48 mg/mL sodium chloride,
1.88 mg/mL
sodium phosphate dibasic heptahydrate, 0.41 mg/mL sodium phosphate monobasic
monohydrate, and Water for Injection, USP.
Date Recue/Date Received 2021-08-12
100741 Cyclophosphamide (commercially available as Cyclophosphamide Capsules,
for oral
use; or Cyclophosphamide Tablets, USP): Cyclophosphamide is a synthetic
antineoplastic
drug chemically related to the nitrogen mustards. The chemical name for
cyclophosphamide
is 2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide
monohydrate
and has the molecular formula C7H15C12N20213.1-120 and a molecular weight of
279.1.
Each capsule for oral use contains 25 mg or 50 mg cyclophosphamide (anhydrous,
USP).
100751 Stereotactic body radiation therapy (SBRT): SBRT has emerged as a safe
and
effective alternative to conventionally-fractionated external beam radiation.
SBRT is a highly
conformal external beam radiation technique, capable of precisely delivering
ablative doses
of radiation in a limited number of fractions. Preclinical data suggest
relatively large doses
per fraction (6-8 Gy) can induce immune responses to tumor antigens. The steep
dose fall-off
observed with SBRT treatments allows high doses per fraction to be achieved
with limited
radiation exposure to adjacent critical structures.
100761 Most typically patients will receive 4 fractions of radiation per
feasible tumor site
(maximum of 5 sites), at a dose of up to 8 Gy per fraction. If organ at risk
(OAR) dose
constraints cannot be achieved, a dose reduction to 6 Gy per fraction can be
performed at the
discretion of the treating physician. Radiation treatments will be
administered twice every 21
days for the first 2 treatment cycles. A single treatment plan will be devised
for each lesion
prior to initiation of therapy. Given the length of time between fractions, a
repeat CT
simulation and adjustments to the treatment plan may be performed at the
discretion of the
radiation oncologist if significant tumor regression (as noted
radiographically or by clinical
exam) occurs between fractions. Changes to the treatment plan should only be
made to
exclude normal tissues or critical structures that are clearly uninvolved by
tumor and which
may have fallen into the G'TV as a result of tumor regression.
100771 Radiation dose will be prescribed such that 95% of the PTV receives the
prescription
dose or greater, though reductions to as low as 80% coverage will be
considered acceptable if
deemed appropriate by the treating physician in order to spare critical normal
structures; in
such cases, the region receiving less than 95% of the prescription dose should
be limited to
the periphery of the PTV and outside of the GTV. A high degree of dose
heterogeneity is to
be expected with SBRT. As such a central "hotspot" is expected, and the
prescription dose
should be within 60 90% of the maximum dose within the PTV. Radiation dose
calculations
will be performed using tissue heterogeneity corrections
21
Date Recue/Date Received 2021-08-12
100781 While not limiting to the inventive subject matter, contemplated
pharmaceutical
agents and radiation will be administered following the exemplary dosages
listed in Table 2.
Of course, it should be appreciated that patient and disease specific factors
(e.g., gender,
weight, disease response or progression. adverse reactions, etc.) may dictate
a change in the
particular dosage and schedule.
Drugs Dosage Mode of Administration
Aldoxorubicin HCI 80 mg/m2 IV aver approximately 30
minutes
ALT-803 10 pg/kg SC
Ad5-based vaccines: ETBX- 1 x 1011 VP/vaccine/dose SC
051 (Brachyury) and ETBX-
061 (MUC1)
Yeast-based vaccine: 80 VU/dose SC
GI-6301 (Brachyury)
haNK 2 x i0 cells/dose IV
Avelumab 10 mg/kg IV
Cetuximab 250 mg/m2 IV
Cyclophosphamide 25 mg BID (days 1-5) PO
25 mg daily (days 8-12)
SBRT 8 Gy maximum (exact dose to be External beam
radiation
determined by the radiation oncologist)
Table 2
100791 A typical treatment schema for the induction phase is shown in Figure
8, and a
typical treatment schema for the maintenance phase is shown in Figure 9.
100801 For example, an exemplary treatment regimen for the induction phase is
contemplated, lasting about 8 weeks (minimum) to about 1 year (maximum).
Treatment will
include repeated 3-week cycles for a maximum treatment period of 2 years, as
follows:
22
Date Recue/Date Received 2021-08-12
100811 Days 1 and 8, every 3 weeks: Aldoxorubicin HC1 (80 mg/m2 IV over
approximately
30 minutes).
100821 Days 1-5, every 3 weeks: Cyclophosphamide (25 mg by mouth [P0] twice a
day
[BID]).
100831 Day 5 (1 1 day), every 3 weeks for 3 cycles then every 9 weeks
thereafter: Ad5-based
vaccines: ETBX-051 (Brachyury) and ETBX-061 (MUC1), (1 x 1011 virus particles
[VP]/vaccine/dose subcutaneously [SC]).
100841 Day 8, every 3 weeks: Avelumab (10 mg/kg IV over approximately 1 hour).
100851 Days 8-12, every 3 weeks: Cyclophosphamide (25 mg by mouth [P0] daily).
100861 Days 8 and 15, every 3 weeks: SBRT (not to exceed 8 Gy, exact dose to
be
determined by the radiation oncologist; for the first 2 cycles only).
100871 Day 9, every 3 weeks: ALT-803 (10 pg/kg SC at least 30 minutes prior to
haNK
infusion); haNK (2 x 109 cells/dose IV); Cetuximab (250 mg/m2 IV).
100881 Days 11, every 3 weeks: haNK (2 x 109 cells/dose IV).
100891 Day 11, every 3 weeks for 3 cycles and every 9 weeks thereafter: Yeast-
based
vaccine: GI-6301(Brachyury) (80 yeast units [YU]/dose SC).
100901 Day 16, every 3 weeks: ALT-803 (10 g/kg SC at least 30 minutes prior
to haNK
infusion); haNK (2 x 109 cells/dose IV); Cetuximab (250 mg/m2 IV).
100911 Day 18, every 3 weeks: haNK (2 x 109 cells/dose IV).
100921 An exemplary treatment regimen for the maintenance phase, which may
last up to 1
year following completion of the last treatment in the induction phase will
include repeated
cycles, as follows:
100931 Day 1, every 3 weeks: Avelumab (10 mg/kg TV over approximately 1 hour);
Cetuximab (250 mg/m2 IV); ALT-803 (10 g/kg SC) (at least 30 minutes prior to
haNK
infusion); haNK (2 x 109 cells/dose IV).
23
Date Recue/Date Received 2021-08-12
100941 Day 1, every 9 weeks: Ad5-based vaccines: ETBX-051 (Brachyury) and ETBX-
061
(MUC1) (1 x 1011 VP/vaccine/dose SC); Yeast-based vaccine: GI-6301 (Brachyury)
(80
YU/dose SC), approximately 2 hours after administration of Ad-5 based
vaccines.
[0095] For tumor response evaluation it is contemplated that patients will be
evaluated for
tumor burden by CT and/or MRI imaging at screening (up to 28 days before
treatment).
Subsequent evaluations for tumor response will occur every 8 weeks or 12 weeks
(depending
on time on treatment, as described previously) ( 7 days) following the
administration of the
first treatment. Imaging will continue until PD is documented or the subject
completes study
follow-up. When disease progression per RECIST Version 1.1 is initially
observed, an
imaging assessment will be done 4-6 weeks after the initial PD assessment to
rule out tumor
pseudoprogression. For patients exhibiting a response (PR or CR), a
confirmatory imaging
assessment will be done 4-6 weeks after the initial response. Evaluations may
include CT
and/or MRI scans of the chest, abdomen, pelvis (optional unless known pelvic
disease is
present at baseline), and brain (only as clinically warranted based on
symptoms/findings).
100961 Prior to treatment, tumor lesions to be followed for response will be
clearly identified
by location and selected and categorized as target or non-target lesions.
Target lesions
include those lesions that can be accurately measured in at least 1 dimension
as 10 mm,
using CT, PET-CT, or MRI with a slice thickness < 5 mm. Malignant lymph nodes
with a
short axis diameter > 15 mm can be considered target lesions. Up to a maximum
of 2 target
lesions per organ and 5 target lesions in total will be identified at
baseline. These lesions
should be representative of all involved organs and selected based on their
size (those with
the longest diameter) and their suitability for accurate repeated
measurements. A sum of the
longest lesion diameter (LLD) for all target lesions will be calculated and
reported as the
baseline sum LLD. For malignant lymph nodes identified as target lesions, the
short axis
diameter will be used in the sum of LLD calculation. All other lesions (or
sites of disease)
should be identified as non target lesions (including bone lesions).
100971 All post-baseline response assessments should follow the same lesions
identified at
baseline. The same mode(s) of assessment (e.g., CT or MRI) used to
identify/evaluate lesions
at baseline should be used throughout the course of the study unless subject
safety
necessitates a change (e.g., allergic reaction to contrast media).
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Date Recue/Date Received 2021-08-12
100981 For tumor molecular profiling it is contemplated that genomic
sequencing of tumor
cells from tissue relative to non-tumor cells from whole blood will be
conducted to identify
tumor-specific genomic variances that may contribute to disease progression
and/or response
to treatment. RNA sequencing will be conducted to provide expression data and
give
relevance to DNA mutations. Quantitative proteomics analysis will be conducted
to
determine the absolute amounts of specific proteins, to confirm expression of
genes that are
correlative of disease progression and/or response, and to determine cutoff
values for
response.
100991 Tumor molecular profiling will preferably be performed on FFPE tumor
tissue and
whole blood (subject-matched normal comparator against the tumor tissue) by
next-
generation sequencing and mass spectrometry-based quantitative proteomics.
Tumor tissue
from a biopsy will also be collected 8 weeks after the start of treatment.
Furthermore, if
additional tumor biopsies will be performed, further tumor molecular profiling
will be
performed on those samples, as well.
1001001 For example, tumor tissue and whole blood samples will be collected
and shipped
in accordance with the instruction cards included in a Tissue Specimen Kit and
Blood
Specimen Kit. An FFPE tumor tissue specimen is typically used for the
extraction of tumor
DNA, tumor RNA, and tumor protein. A whole blood sample is typically used for
the
extraction of subject normal DNA. Tumor tissue and whole blood will be
processed in a
CL1A certified and CAP-accredited clinical laboratories (e.g., NantOmics,
LLC.;
ResearchDx, LLC.; and Expression Pathology. Inc. dba OncoPlex Diagnostics).
1001011 Immunology Analysis: Whole blood for immunology analysis will be
collected,
evely 6 weeks in the induction phase and every 8 weeks in the maintenance
phase during
routine blood draws, and at the end of treatment. If a tumor biopsy will be
performed at
screening, blood samples for immunology analysis may be collected prior to the
biopsy.
Blood samples will be stored in a laboratory to be determined. Immune
responses will be
evaluated by standard immune assays. Correlations between therapy-induced
immune
changes and subject outcomes will be assessed.
1001021 Circulating Tumor DNA and RNA Assays: Tumors evolve during therapy,
and
drug-resistant cells emerge, which are difficult to detect and may cause the
tumor to become
resistant to the initial treatment. Blood-based testing for ctDNA and ctRNA
can track the
Date Recue/Date Received 2021-08-12
emergence of drug-resistant tumor cells and can identify new drug targets and
treatment
options for patients. To that end, whole blood for ctDNA/ctRNA analysis will
be collected
during the screening period for subjects who have been enrolled in the study,
every 6 weeks
in the induction phase and every 8 weeks in the maintenance during routine
blood draws, and
at the end of treatment. If a tumor biopsy will be performed at screening,
blood samples for
ctDNA and ctRNA analysis must be collected prior to the biopsy. Expression
levels of
specific tumor- and immune-related analy-tes in ctDNA and ctRNA will be
measured by
qPCR and possibly other methods (e.g., DNA/RNA sequencing) and analyzed for
correlations with subject outcomes.
1001031 As used in the description herein and throughout the claims that
follow, the
meaning of "a," "an," and "the" includes plural reference unless the context
clearly dictates
otherwise. Also, as used in the description herein, the meaning of "in"
includes "in" and
"on" unless the context clearly dictates otherwise. Furthermore, and unless
the context
dictates otherwise, the term "coupled to" is intended to include both direct
coupling (in which
two elements that are coupled to each other contact each other) and indirect
coupling (in
which at least one additional element is located between the two elements).
Therefore, the
terms "coupled to" and "coupled with" are used synonymously. Unless the
context dictates
the contrary, all ranges set forth herein should be interpreted as being
inclusive of their
endpoints, and open-ended ranges should be interpreted to include commercially
practical
values. Similarly, all lists of values should be considered as inclusive of
intermediate values
unless the context indicates the contrary.
1001041 It should be apparent to those skilled in the art that many more
modifications
besides those already described are possible without departing from the
inventive concepts
herein. The inventive subject matter, therefore, is not to be restricted
except in the scope of
the appended claims. Moreover, in interpreting both the specification and the
claims, all
terms should be interpreted in the broadest possible manner consistent with
the context. In
particular, the terms "comprises" and "comprising" should be interpreted as
referring to
elements, components, or steps in a non-exclusive manner, indicating that the
referenced
elements, components, or steps may be present, or utilized, or combined with
other elements,
components, or steps that are not expressly referenced. Where the
specification claims refers
to at least one of something selected from the group consisting of A, B, C
.... and N, the text
26
Date Recue/Date Received 2021-08-12
should be interpreted as requiring only one element from the group, not A plus
N, or B plus
N, etc.
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Date Recue/Date Received 2021-08-12
